JPH0365198A - Production of d-pantolactone - Google Patents

Abstract

PURPOSE:To obtain high optical purity in a short time by separating D-pantoic acid derived from asymmetric hydrolysis of only D-form of D,L-pantolactone with a specific microorganism followed by the transforming. CONSTITUTION:A microorganism (A) having lactone hydrolyzing ability is selected from microorganism belonging to genuses such as Fusarium, Cylindrocarpon, Gibberella, Aspergillus, Penicillium, Rhizopus, Volutella, Gliocladium, Eurotium, Nectria, Schizophyllum, Myrothecium and Neurospora. Next, the A-strain is aerobically cultured in a medium containing glucose and peptone, etc., at pH3-9 and at 10-50 deg.C for 1-7 days to obtain a cultured substance (B). Then, the B-component is added to a solution of D,L-pantolactone having 10-500g/l concentration and reacted at pH3-8 and at 10-50 deg.C for several hours to 3 days to obtain a reaction product (C). Thus, the C-component is separated and resultant D-pantoic acid is heated and extracted under acidic condition to recover D-pantolactone.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a novel method for producing monopantolactone, which is useful as a medically or physiologically important vitamin and is useful as an intermediate in the production of monopantothenic acid and pantethine. .

[Prior art] Conventionally, D-pantolactone was synthesized chemically or
-Produced by optical resolution of pantolactone.

However, this method requires expensive resolving agents such as quinine and brucine, and has drawbacks such as difficulty in recovering D-pantolactone.

On the other hand, the following methods have been reported to date as enzymatic resolution methods for D,L-pantolactone.

In other words, the method described in Japanese Patent Publication No. 47-19745 uses microorganisms to completely decompose the divantolactone in b,1-pantolactone to obtain only D-pantolactone. However, with this method, D
, has the disadvantage that half of the L-pantolactone is lost.

The method described in JP-A No. 61-293386 is
Using microorganisms, oxidize only pantolactone in D, L-pantolactone to getobantolactone,
This method is further asymmetrically reduced to give D-pantolactone, but since both the substrate concentration and the reaction rate are low, it is of little significance as a practical method.

JP-A-57-152895 and JP-A-62-294
The method described in each publication of No. 092 is a method of selectively asymmetrically hydrolyzing the L-unit in D,L-pantolactone using microorganisms to obtain D-pantolactone.
Failure to completely hydrolyze L-unit has the disadvantage that D-pantolactone with high optical purity cannot be obtained, and both substrate concentration and reaction rate are low;
These too cannot be practical methods.

[Disclosure of the Invention] As a result of extensive research into the asymmetric hydrolysis of D,L-pantolactone, the present inventors have found that by using specific microorganisms, only D in D,L-pantolactone can be isolated. By selectively asymmetrically hydrolyzing , D-pantoic acid is produced, and the D-pantoic acid is separated and converted to D-pantolactone, thereby efficiently converting D-pantolactone from 0, seabantolactone. The present invention, which has discovered that lactones can be obtained, was made based on this knowledge.

That is, the present invention relates to the genus Fusarium, Cylindrocarpon, Gibberella, Asbergilus, Penicillium, Rhizopus, Volterra, Gliocladium, Euroteium, Nectria, Schizophyllum, Myrocesium, and Neurospora. , a microorganism having the ability to hydrolyze lactone selected from microorganisms belonging to the genus Acrimonium, genus Tuberculina, genus Abcisia, genus Sporothrix, genus Verteicillium, or genus Arthroderma is used, and D-units in L-pantolactone are selectively removed. The present invention provides a method for producing D-pantolactone, which comprises producing D-pantoic acid by asymmetric hydrolysis, separating the D-pantoic acid, and converting it to D-pantolactone. be.

The present invention has the following advantages: compared to the method of selectively asymmetrically hydrolyzing monomers in L-pantolactone, the substrate concentration can be increased, the reaction time can be set short, and the optical purity can be extremely high. It has many advantages such as being able to obtain a high amount of D-pantolactone.

The present invention will be explained in detail below.

The present inventors inoculated seed bacteria from a slant medium into various liquid media (5 tags), cultured them aerobically with shaking at 28°C for 2 to 7 days, and then obtained bacterial bodies by centrifugation or r-filtration. 01L-Pantora 21fu 2% concentration of 0.2HTri on the body
s-HCj *ii! 5i2- was added and shaken overnight at 28°C, and the resulting reaction solution was measured for the amount of vantolactone decreased and the amount of pantoic acid produced using HPLC, and the optical purity of vantolactone was measured using GLC. did.

As a result, 0. Microorganisms that selectively asymmetrically hydrolyze seabantlactone include Fusarium, Cylindrocarpon, Gibberella, Aspergillus, Penicillium, Rhizopus, Volterra, Gliocladium, and Euroteium. , Nectria, Schizophyllum, Myrocesium, Neurospora, Acrimonium, Tuberculina, Absisia, Sporothrix, Verteicillium, or Arthroderma are used industrially to produce D-pantolactone. It was discovered that the microorganism is suitable for the production of

Among the microorganisms belonging to each of the above genera, some are found to have particularly excellent D-main selective asymmetric hydrolyzing ability.

In the method of the present invention, various conditions for culturing microorganisms vary depending on the strain used, but the culture medium includes carbohydrates such as glucose and sucrose, alcohols such as ethanol 7 and glycerol, and oleic acid as a carbon source. , fatty acids and their esters such as stearic acid, oils such as rapeseed oil and soybean oil, nitrogen sources such as ammonium sulfate, sodium nitrate, peptone, casamino acids, cornstarch liquor, bran, yeast extract, and inorganic salts such as sulfuric acid. A medium containing malt extract, meat extract, etc. as other nutritional sources such as magnesium, sodium chloride, calcium carbonate, potassium hydrogen phosphate, and potassium trihydrogen phosphate is used. Cultivation is carried out aerobically, usually for about 1 to 7 days, at a culture medium DH of 3 to 9, and at a culture temperature of 10 to 50°C.

In the method of the present invention, the microorganism used is a culture obtained by culturing a bacterial strain in a liquid medium, a bacterial cell isolated from a culture solution, a dried bacterial cell obtained by processing a bacterial cell or a culture, or a fixed bacterial cell. Any form such as a transformed bacterial cell can be used.

The operation can be carried out either quarterly, semi-batchwise or continuously. The concentration of 0,thi-pantolactone used is usually 10- DH of 0 reaction system which is 3 days from time
is usually about 3 to 8.

As a result of D-mono-selective asymmetric hydrolysis of L-pantolactone by microorganisms, D-pantoic acid is produced, the pH of the reaction solution decreases, and the reaction rate also decreases. In order to increase the reaction rate, It is preferable to maintain the pH of the reaction solution at the optimum pH for the lactone hydrolase of each microorganism. In this case, an alkali metal or alkaline earth metal hydroxide or carbonate is used as an inorganic base to maintain 11H. In addition, ammonia water, etc. are used.

After the reaction is complete, the D-bantolactone in the reaction solution that has not undergone hydrolysis is separated by extraction with an organic solvent.Next, the D-bantoic acid remaining in the reaction solution is heated under acidic conditions. After that, it is converted to D-pantolactone.

The D-pantolactone produced is recovered by extraction using an organic solvent. It can be used as a raw material for the invention method.

The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples.

Examples 1-19 Glucose 1%, peptone 0.5%, yeast extract 0.5
%, and 5 ml of a liquid medium (pH 6.5) consisting of 0.5% corn stay glycerin in a test tube. The mixture was divided into portions and sterilized by heating at 121°C for 20 minutes in an autoclave.

The various strains listed in Table 1 were inoculated from the slant medium into the culture medium in each test tube, and cultured aerobically with shaking at 28° C. for 5 days.

Obtain bacterial cells by culture medium Itf&, filtration, and each of these vM
For each container containing #, D, L-pantolactone 2
% concentration of 0.2H chloride-HCj M* solution 2 ra
After 6 reactions, the cells were added at 28° C. (pi 7.5) and shaken overnight at 28° C., and the bacterial cells were removed by filtration.
(Nucleosil 5C+*φ4,6 xj 15
0 ram, solution M solution 10% methanol, flow rate 1 mg/n
After extracting and separating unreacted pantolactone in the reaction solution using ethyl acetate, the amount of decrease in pantolactone and the amount of pantoic acid produced in each reaction solution were measured by The remaining pantoic acid was heated under acidic conditions with hydrochloric acid to effect lactonization, and the resulting D-pantolactone was extracted using ethyl acetate. The optical purity of D-pantolactone obtained from this pantoic acid was measured by GLC (Analytica
l Biochenistry, 1129-19 (1
981) ). The results are shown in Table 1.

1 Table (continued) Note: IFONG indicates the Fermentation Research Institute catalog number.

Examples 20-23 Glycerol 2%, Peltone 0.5%, yeast extract 0.
5% and 0.5% corn staple liquor (ptl 5.5) 500I with 10011j
Ill! Using a shake flask, each strain listed in Table 2 was incubated aerobically at 28°C for 6 days with JIi!
It was cultured. After culturing, the bacterial cells are obtained by filtration, each of these bacterial cells is stored in a container, and each container is filled with
A 30% D, L-pantolactone aqueous solution (25Ilj) was added, and while stirring, 28% ammonia water was added dropwise to the reaction solution while maintaining l at 6.5 to 7.5, and the reaction was carried out at 28°C for 2 days. Ta. In the same manner as in Examples 1 to 19,
Post-treatment was performed after the reaction. Yield, yield, and [α]% of the obtained D-pantolactone and the recovered monopantolactone. are shown in Table 2.

Claims (1)

[Claims] (1) Fusarium, Cylindrocarpon, Gibberella, Aspergillus, Penicillium, Rhizopus,
Microorganisms selected from the genus Volterra, Gliocladium, Euroteium, Nectria, Schizophyllum, Myrocesium, Neurospora, Acrimonium, Tuberculina, Absisia, Sporothrix, Verteicillium or Arthroderma. D-pantoic acid is produced by selectively asymmetrically hydrolyzing only the D-isomer in D,L-pantolactone using a microorganism capable of hydrolyzing lactone, and the D-pantoic acid is separated. A method for producing D-pantolactone, which comprises converting it into D-pantolactone.